1. Field of the Invention
This invention relates to a monoclonal antibody against a plasminogen-activating factor produced by the culture of fibroblasts derived from human fetal lung tissue. More particularly, it relates to a monoclonal antibody against the non-urokinase type plasminogen-activating factor obtained by culturing the aforesaid fibroblasts to cause the concurrent production of a urokinase type plasminogen-activating factor and a plasminogen-activating factor having properties different from those of the urokinase type plasminogen-activating factor, and then removing the urokinase type plasminogen-activating factor selectively from the culture medium containing both plasminogen-activating factors.
2. Description of the Prior Art
In the treatment of thromboembolic disorders, urokinase isolated from human urine or a culture of kidney cell, and streptokinase isolated from the filtrate of a culture of .beta.-hemolytic streptococci are now being used as plasminogen activators for the lysis of fibrin.
However, urokinase has a low affinity for fibrin and produces side effects such as hemorrhage due to high-dose administration. On the other hand, streptokinase is an enzyme protein derived from a microorganism and, when administered to human beings, has the possibility of causing allergy. Accordingly, it cannot be positively said that no problem is encountered in using these plasminogen activators for the treatment of thromboembolic disorders.
In recent years, attention has been focused on tissue type plasminogen activators which, unlike the aforesaid plasminogen activators, are found in tissues of the living organisms.
One such tissue type plasminogen activator was isolated from the cell-free culture medium of human melanoma cells and purified by Rijken and Collen [J.B.C., 256, 7035-7041(1981); Thromb. Haemostas. (Stuttgart), 48, 294-296(1982)], and it was distinguished by its molecular weight from the aforesaid urokinase and streptokinase. Thereafter, as regards this tissue type plasminogen activator derived from human melanoma cells, Pennica et al. [Nature, 301, 214-221(1983)] determined its amino acid sequence defining the primary structure of its molecule, and Bennet [Thromb. Haemostas., 50, 106(1983)] demonstrated that there are two variants having the sugar chain attached to different positions, and that their molecular weights differ from each other by 3000.
As other tissue type plasminogen activators, Vetterlain et al. [J.B.C., 254, 575-578(1979); J.B.C., 255, 3665-3672(1980)] have reported the plasminogen activators produced by normal fibroblast strain IMR-90 derived from human fetal lung tissue, and have shown that these plasminogen activators include a urokinase type plasminogen activator having a molecular weight of 50,000 to 60,000 and a new type of plasminogen activator having a molecular weight of 73,000 and incapable of being neutralized with anti-urokinase antibodies.
Moreover, Wilson et al. [Cancer Research, 40, 933-938(1980)] have also confirmed that fibroblasts derived from the lung tissue of an 8-weeks-old human fetus produce a urokinase type plasminogen activator having a molecular weight of 60,000 and a new type of plasminogen activator having a molecular weight of about 70,000 and incapable of being neutralized with anti-urokinase antibodies.
On the other hand, there has recently been published an invention relating to the preparation of a monoclonal antibody of the aforesaid tissue type plasminogen activator derived from human melanoma cells (Japanese Patent Laid-Open No. 5121/'84). In this invention, mouse myeloma cells were fused with spleen cells from a mouse immunized with the tissue type plasminogen activator derived from human melanoma cells. Thus, there were obtained 40 hybridomas capable of producing an antibody to the aforesaid plasminogen activator. It is shown, however, that the antibodies produced by only 5 hybridomas were specific for the aforesaid plasminogen activator and the antibodies produced by most of the other hybridomas exhibited a cross reaction with both the aforesaid plasminogen activator and urokinase. According to the teachings of the invention, this is not attributable to the fact that the aforesaid tissue type plasminogen activator used as the antigen was contaminated with urokinase, but to the fact that the aforesaid plasminogen and urokinase are cross reacting materials, i.e., these two enzymes have common antigenic determinants.
The method for isolating and purifying the aforesaid new type of plasminogen activator produced by normal fibroblasts derived from human fetal lung tissue has not yet been established. Consequently, its immunochemical properties and primary structure have not been elucidated.
Accordingly, it is not yet clear whether or not the aforesaid plasminogen activator produced by normal fibroblasts derived from human fetal lung tissue is the same substance as the aforesaid tissue type plasminogen activator derived from human melanoma cells, and whether or not their molecules have different primary structures.
In this connection, there has recently been proposed a method for isolating the aforesaid new type of plasminogen activator derived from human normal fibroblasts by using a zinc chelate column, fibrin column, concanavalin A-Sepharose column, arginine-Sepharose column or the like (Japanese Patent Laid-Open No. 51220/'84).
In this method, however, such columns fail to bind the aforesaid plasminogen activator specifically, so that it is impossible to obtain the plasminogen activator in a highly pure form and in a high recovery. Thus, this method lacks practical utility.
Moreover, a purification method using a specific antibody obtained from an antiserum against the aforesaid plasminogen activator is also conceivable. However, since such an antibody is a mixture, this method has the disadvantage that the titer of the antibody per unit weight of protein is low, its affinity for the plasminogen activator and its stability are not uniform, and the column chromatography cannot be carried out under consistent conditions. For these reasons, the efficiency of the adsorption to the column is low and the recovery of the plasminogen activator is not satisfactorily high.